N(6)-methyladenosine-mediated miR-380-3p maturation and upregulation promotes cancer aggressiveness in pancreatic cancer

ABSTRACT N(6)-methyladenosine (m6A)-modified microRNAs (miRNAs) are relevant to cancer progression. Also, although the involvement of miR-380-3p in regulating cancer progression in bladder cancer and neuroblastoma has been preliminarily explored, its role in other types of cancer, such as pancreatic cancer (PC), has not been studied. Thus, this study aimed to investigate the role of miR-380-3p in regulating PC progression. Here, through performing Real-Time qPCR, we evidenced that miR-380-3p was significantly upregulated in the clinical pancreatic cancer tissues and cells compared to their normal counterparts. Interestingly, miR-380-3p was enriched with m6A modifications, and elimination of m6A modifications by deleting METTL3 and METTL14 synergistically suppressed miR-380-3p expressions in PC cells. Next, the gain and loss-of-function experiments verified that knockdown of miR-380-3p suppressed cell proliferation, epithelial–mesenchymal transition (EMT), and tumorigenesis in PC cells in vitro and in vivo, whereas miR-380-3p overexpression had opposite effects. Furthermore, the underlying mechanisms were uncovered, and our data suggested that miR-380-3p targeted the 3’ untranslated regions (3ʹUTRs) of PTEN for its inhibition and degradation, resulting in the activation of the downstream Akt signal pathway. Moreover, the rescuing experiments validated that both PTEN overexpression and Akt pathway inhibitor LY294002 abrogated the promoting effects of miR-380-3p overexpression on cancer aggressiveness in PC cells. Collectively, this study firstly investigated the role of the m6A-associated miR-380-3p/PTEN/Akt pathway in regulating PC progression, which provided novel therapeutic and diagnostic biomarkers for this cancer.


Introduction
As one of the most lethal malignant cancers that ranks fourth in cancer-related deaths worldwide, pancreatic cancer (PC) seriously endangers the life of human beings [1,2]. Although the therapeutic technologies for cancer treatment have been dramatically developed in the past decades, the prognosis of PC patients is still very poor, and the 5-year survival rate for PC patients is less than 5% [1,2]. According to previous literatures, the poor prognosis of PC is mostly attributed to early metastasis of PC cells into the lymph nodes [3,4], but the detailed mechanisms are still not fully understood, which makes blockage of this process impossible at early stage of PC pathogenesis. As one of the most important malignant phenotypes, epithelial-mesenchymal transition (EMT) is a process that transforms the original epithelial phenotypes of cancer cells into mesenchymal phenotypes, and the EMT-associated biomarkers (N-cadherin, Vimentin, and E-cadherin) are significantly altered during this process [5,6]. EMT plays a critical role in facilitating cancer metastasis in multiple cancers, including PC [7,8]. Specifically, data from different teams point out that inhibition of EMT is effective to hamper the metastasis of PC cells [7,9]. Thus, identification of the novel regulators for EMT may shed light on the discovery of effective treatment strategies for PC treatment. N(6)-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic RNAs, which plays a critical role in regulating the fate of the associated RNAs [10][11][12]. Mechanistically, the m6A modifications are introduced by 'Writers,' including methyltransferase-like 3 (METTL3), METTL4, and Wilms tumor 1-associated protein, and those m6A modifications are removed and demethylated by 'Erasers,' such as human AlkB homolog H5 (ALKBH5) and fat mass and obesity-associated protein (FTO) [10][11][12]. Interestingly, m6A modifications are recently reported to participate in the regulation of various diseases, especially in cancers [13,14]. For example, Zhang et al. report that IGF2BP1 m6Adependently stabilizes PEG10 mRNA to promote endometrial cancer progression [14], and ALKBH5-HOXA10 loop-mediated JAK2 m6A demethylation contributes to cisplatin-resistance in epithelial ovarian cancer [13]. Notably, m6A methylation is considered as a critical biological process that affects the development of PC [15][16][17]. As previously described, various biological functions, including cell proliferation [18,19], migration [18,19] and EMT [20,21], can all be regulated by m6A methylation. Especially, m6A modifications influence EMT process, and Liu et al. describe that RNA m6A methylation regulates EMT of cancer cells and Snail translation [21], and Yue et al. find that METTL3-mediated m6A modification is critical for EMT and metastasis of gastric cancer [20].
Here, we focused on investigating the involvement and underlying mechanisms by which the m6A/miR-380-3p/PTEN/Akt pathway regulated cancer aggressiveness in PC. Our study will broaden our knowledge in this field, and may provide novel biomarkers for PC diagnosis, treatment, and prognosis.

Clinical specimens
The adjacent normal pancreatic tissues (N = 32) and cancerous PC tissues (N = 32) were collected from PC patients by surgical resection, and the clinical specimens were confirmed by two experienced pathologists in our hospital. The clinical tissues were stored at −70°C conditions for further utilization. The clinical experiments were approved by the Ethics Committee affiliated to the Second Hospital of Tianjin Medical University KY2022K167), and the informed consent forms had been obtained from all the participants.

Cell culture, vectors delivery, and treatment
The normal human pancreatic ductal endothelial cell (HPDE) and PC cell lines (PANC1, Capan-2, SW1990, AsPC-1, and BXPC-3) were bought from American Type Culture Collection (ATCC, VA, USA). The PC cells were maintained in the Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) with high-glucose, and the normal HPDE cells were cultured in the RPMI 1640 medium (Gibco, USA). All of the above two mediums were supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and the cells were maintained in the incubator with the following culture conditions: 5% CO 2 humidified air at 37°C. The cells were subsequently transfected with miR-380-3p mimic/inhibitor (Sangon Biotech, Shanghai, China) and PTEN overexpression/downregulation vectors (Sangon Biotech, Shanghai, China) by using the Lipofectamine TM 3000 reagent (Thermo Fisher Scientific, MA, USA) in keeping with the manufacturer's protocol.

Real-Time qPCR analysis
The mRNA levels of the genes were examined by performing the Real-Time qPCR analysis in accordance with the experimental procedures provided by the previous publications. In brief, the total RNA was extracted from the PC tissues and cells by using the Trizol reagent (Invitrogen, USA). Reverse transcription was achieved by the SuperScriptTM II reverse transcriptase (Invitrogen, USA), and the RNA levels of CDK2, CDK6, Cyclin D1, GAPDH, miR-380-3p, and U6 were determined by using the SYBR Green qPCR system (Takara, Japan). The primer sequences for the associated genes are listed in Table 1.

Western blot analysis
The PC tissues and cells were prepared and lysed by radioimmunoprecipitation lysis buffer (RIPA, Beyotime, Shanghai, China), and protein concentration was determined by BCA kit (Thermo Fisher Scientific, USA). Proteins were subsequently separated by 10% SDS-PAGE based on their molecular weight, and transferred onto the PVDF membranes (Millipore, MA, USA), which were further blocked by 5% slim milk, and probed with the primary antibodies against E-cadherin (Abcam, UK), N-cadherin (Abcam, UK), Vimentin (Abcam, UK), GAPDH (Abcam, UK), PTEN (Abcam, UK), Akt (Abcam, UK), and p-Akt (Abcam, UK) at 4°C overnight. The membranes were then washed by PBS buffer and incubated with the HRP-conjugated secondary antibodies (Abcam, UK) for 2 h at room temperature. The ECL system (BioRad, USA) was employed to visualize the protein bands, and the gray values were calculated to represent relative expression levels of the target proteins.

Cell counting kit-8 (CCK-8) assay
The PC cell lines PANC1 and SW1990 were cultured in the 96-well plates at a density of 5,000 cells per well, and 10 μl CCK-8 reaction solution (Dojindo, Japan) was added to each well for 2 h in the incubator with standard culture conditions. The microplate reader (Thermo Fisher Scientific, USA) was used to examine the optical density (OD) values at the absorbance of 450 nm, which represented the relative cell proliferation abilities of the cells.

Methylated RNA immunoprecipitation (Me-RIP) assay
TRIzol reagent (Invitrogen, USA) was used to extract total RNA, which were, respectively, incubated with the magnetic beads binding with anti-m6A and anti-immunoglobulin antibodies, and m6A modified RNAs were washed with elution buffer to obtain the associated miRNAs. Then, Real-Time qPCR analysis was performed to examine miR-380-3p enrichment in the PC cells.

Transwell assay
The PC cells were cultured in the upper chamber with FBS-free culture medium of the transwell plates purchased from Costar (USA), and the lower chamber was supplemented with 10% FBS. At 24 hours post-culture, the cells were washed 3 times with PBS buffer, fixed by 4% paraformaldehyde and stained with 0.1% crystal violet. The cell numbers were counted under a light microscope (ThermoFisher, USA).

Colony formation assay
The PC cells were seeded onto the 6-well plates and were grown for 14 days. Then, the cells were fixed by 4% paraformaldehyde and were stained with 0.1% crystal violet. A light microscope (ThermoFisher, USA) was employed to count the colony numbers.

Animal experiments
The female BALB/c nude mice (6-week-old) were purchased and fed in the Animal Center Affiliated to Tianjin Medical University in the specificpathogen-free (SPF) conditions. The PC cell line PANC1 was subcutaneously injected into the dorsal flank of the mice at the concentration of 1 × 10 6 cells per mouse. At 4 weeks postinjection, the mice were anesthetized by using the pentobarbital sodium injection method, and the mice were sacrificed by taking off the neck method. The tumors were surgically obtained, weighed, and the tumor volume was measured.
The animal experiments were approved by the Ethics Committee of Tianjin Medical University (TMUaMEC2022059).

Dual luciferase reporter gene system assay
The targeting sites between miR-380-3p and PTEN mRNA were predicted by performing the online starBase software, and the binding sites in PTEN were mutated (Mut-PTEN). Then, the wild-type PTEN (WT-PTEN) and Mut-PTEN sequences were cloned into the downstream of the luciferase gene of the psiCHECK2 vectors (Promega, MI, USA), and the luciferase plasmids were cotransfected with the miR-NC, miR-380-3p mimic/inhibitor into the PC cell line PANC1 and SW1990 by using the Lipofectamine TM 3000 reagent (Thermo Fisher Scientific, MA, USA). At 48 h post-culture, the cells were lysed with Passive Lysis buffer (Promega, MI, USA), and a Dual-Luciferase Reporter Assay System (Promega, MI, USA) was employed to detect the relative luciferase activities.

Data collection and analysis
Data are presented as Means ± Standard Deviations and analyzed by using the SPSS 18.0 software. Patients' survival was determined by performing the Kaplan-Meier Survival analysis with Log-Rank test. The Student's t-test was employed to compare the means from two groups, and means from multiple groups were compared by using the one-way ANOVA analysis. We set the parameter of P < 0.05 as statistical significance, which were indicated as '*' in the whole manuscript.

M6A-mediated upregulation of miR-380-3p is relevant to cancer progression in PC
Although the involvement of miR-380-3p in regulating cancer aggressiveness in bladder cancer [28] and neuroblastoma [29,30] has been preliminarily investigated, the tumor regulating effects and underlying mechanisms of this miRNA in regulating PC development have not been discussed. To explore this issue, in this study, the cancerous and non-cancerous tissues were collected from PC patients, and Real-Time qPCR analysis confirmed that miR-380-3p was significantly upregulated in the PC tissues compared to the adjacent normal tissues (Figure 1(a)), which were supported by the data from TCGA dataset in pancreatic adenocarcinoma (PAAD) ( Figure S3). Of note, through conducting Kaplan-Meier survival analysis, we noticed that PC patients with high-expressed miR-380-3p tended to have a worse prognosis (Figure 1(b)). Moreover, clinical analysis results suggested that PC patients with high-expressed miR-380-3p were featured with higher tumor stages, and the expression levels of miR-380-3p had nothing to do with other clinical features, such as age and gender ( Table 2), indicating that miR-380-3p might be a prognostic marker for PC. In addition, the following in vitro experiments confirmed that miR-380-3p was significantly upregulated in the PC cell lines (PANC1, Capan-2, SW1990, AsPC-1, and BXPC-3) compared to the normal HPDE cells (Figure 1(c)). Given that miR-380-3p was especially enriched in the PANC1 and SW1990 cells (Figure 1(c)), these two cell lines were chosen for the following functional experiments. As previously described, m6A modifications participate in the regulation of cancer progression [13,14], and we evidenced that the m6A-associated regulators, including METTL3 ( Figure S2A), METTL14 ( Figure S2B), and FTO ( Figure S2C), were all aberrantly upregulated in the PC cell lines compared to the normal HPDE cells. Also, m6A modifications play a critical role in regulating miRNA splicing and maturation, but it is still unclear whether miR-380-3p is regulated by m6A modifications [34,35]. As shown in Figure S2D-E, our Me-RIP assay confirmed that miR-380-3p was significantly enriched by m6A probes, indicating that miR-380-3p was highly modified by epigenetic m6A markers. Then, the knockdown vectors for m6A writers (METTL3 and METTL14) were, respectively, delivered into the PC cells (Figure 1(d-e)), and we surprisingly noticed that deletion of METTL3 and METTL14 synergistically decreased the expression levels of miR-380-3p in the PC cells (Figure 1(f-g)), suggesting that m6A modifications were crucial for sustaining miR-380-3p expression levels in the PC cells.

MiR-380-3p promotes cell proliferation, migration, and EMT in pancreatic cancer
Since miR-380-3p is verified to be closely associated with PC progression, we then verified its detailed tumor-regulating effects and biological functions by performing the following functional experiments. Specifically, the mimics and inhibitors for miR-380-3p were delivered into the PANC1 and SW1990 cells, and the vectors transfection efficiency was determined by Real-Time qPCR ( Figure S1A, B). Then, through performing CCK-8 assay, we evidenced that overexpressed miR-380-3p promotes cell proliferation abilities in the PC cells, whereas miR-380-3p ablation had opposite effects (Figure 2(a-b)), which were supported by the following experiments that miR-380-3p promoted colony formation abilities in the PC cells ( Figure S4A, B). The above results were supported by the following experiments that miR-380-3p positively regulated the expression levels of cell division associated biomarkers, including CDK2, CDK6, and Cyclin D1 in the PC cells (Figure 2  (c-d)). We also verified that miR-380-3p suppressed E-cadherin but increased the expression levels of N-cadherin and Vimentin to facilitate epithelial-mesenchymal transition (EMT) process in the PC cells (Figure 2(e-g)), and Transwell assay results confirmed that miR-380-3p acted as an oncogene to facilitate cell migration in the PC cells ( Figure S4C, D). Furthermore, the PANC1 cells with miR-380-3p overexpression and downregulation were injected into the dorsal flank of the nude mice, and the results showed that miR-380-3p facilitated tumorigenesis of this PC cells in vivo (Figure 2(h-j)). Consistently, the immunohistochemistry (IHC) assay results confirmed that miR-380-3p positively regulated Ki67 expressions in mice tumor tissues (Figure 2(i)). The above data suggested that miR-380-3p acted as an oncogene to aggravate cancer aggressiveness in PC.

PTEN is confirmed as the downstream target of miR-380-3p
It is reported that miRNAs exert their biological functions through targeting the 3' untranslated regions (3ʹUTRs) of the downstream cancerassociated genes, which causes those genes degradation [38][39][40]. Thus, to determine the possible targets for miR-380-3p, we performed the online starBase software, and found that one critical tumor suppressor, PTEN, was possibly targeted by miR-380-3p, and the targeting sites had been illustrated in Figure 3(a). To confirm the targeting relationship of these two genes, the dual-luciferase reporter gene system assay was conducted, and we illustrated that miR-380-3p mimic significantly suppressed luciferase activities in the PC cells cotransfected with wild-type PTEN luciferase vectors, and the luciferase activities were enforced by the miR-380-3p inhibitor (Figure 3(b-c)), implying that miR-380-3p was capable of targeting the predicting sites. In addition, as shown in Figure 3 (d-f), the overexpression of miR-380-3p decreased the mRNA and protein levels of PTEN in the PC cells, which were promoted by silencing miR-380-3p. The above data supported the notion that miR-380-3p negatively regulated PTEN expressions through targeting its 3' UTR.

Overexpression of miR-380-3p activated the Akt pathway through degrading PTEN
PTEN has been widely reported as a tumor suppressor in multiple cancers, including PC [38][39][40], and it is well recognized as the negative regulator for the classical tumor-promoting Akt pathway [41,42]. However, it is still largely unknown whether miR-380-3p regulated the Akt pathway in a PTENdependent manner. To investigate this issue, the miR-380-3p mimics and inhibitors, and PTEN overexpression (OE-PTEN) and downregulation (KD-PTEN) vectors were, respectively, delivered into the PC cells, and the cells were grouped as follows: Control, miR-mimic group, miR-inhibitor group, miR-mimic + OE-PTEN group, and miR-inhibitor + KD-PTEN group. The Western Blot analysis verified that miR-380-3p positively regulated the expression levels of Akt and phosphorylated Akt (p-Akt) to activate the Akt pathway in the PC cells (Figure 4(a-b)). Of note, the promoting effects of miR-380-3p overexpression on Akt pathway activation were abrogated by upregulating PTEN, and conversely, PTEN ablation re-activated the Akt pathway in the PC cells co-transfected with miR-380-3p inhibitor (Figure 4(a-b)). Those data hinted that miR-380-3p activated the Akt pathway in the PC cell by degrading PTEN.

Upregulated miR-380-3p facilitates pancreatic cancer progression through modulating the PTEN-Akt signal pathway
To ask whether miR-380-3p exerts its tumorpromoting effects in PC through regulating its downstream PTEN-Akt pathway, the PC cells were, respectively, treated with miR-380-3p mimic (miR-mimic), PTEN overexpression vectors (OE-PTEN), and the inhibitor for the Akt pathway (LY294002). The cells were divided into groups as follows: Control, miR-mimic group, miR-mimic + OE-PTEN group, and miR-mimic + LY294002 groups. As expected, the CCK-8 assay confirmed that both PTEN overexpression and LY294002 abrogated the promoting effects of miR-380-3p on cell proliferation abilities ( Figure 5(a-b)). In addition, miR-380-3p overexpression-induced upregulation of CDK2, CDK6, and Cyclin D1 were also abrogated by PTEN overexpression and LY294002 co-treatment ( Figure  5(c-d)). Furthermore, by performing Western Blot analysis, we confirmed that the effects of miR-380-3p overexpression on the EMT-associated biomarkers (E-cadherin, N-cadherin, and Vimentin) were all abolished by PTEN upregulation and LY294002 ( Figure 5(e-g)). Those data hinted that miR-380-3p regulated the PTEN-Akt pathway to exert its tumor-promoting functions in the PC cells.

Discussion
MicroRNAs (miRNAs) are considered as critical diagnostical and prognostic biomarkers for PC [22,23], although the involvement of a novel miR-380-3p in regulating cancer aggressiveness in bladder cancer [28], neuroblastoma [29,30], and breast cancer [31] has been preliminarily investigated, the regulating effects and molecular mechanisms of this miRNA in regulating PC progression have not been reported. In this study, through performing the clinical and preclinical experiments, we evidenced that miR-380-3p was significantly upregulated in the cancerous PC tissues and cells, instead of their non-cancerous counterparts. Also, through conducting the Kaplan-Meier survival analysis, we evidenced that PC patients with high-expressed miR-380-3p had an unfavorable prognosis; the above data was supported by previous publications on other types of cancer, suggesting that aberrant expression of miR-380-3p was closely associated with PC progression and prognosis. In addition, N(6)methyladenosine (m6A) methylation is critical for promoting miRNAs maturation and upregulation [34,35], but it is still unknown whether miR-380-3p can also be controlled by m6A methylation. Our study firstly resolved this problem and evidenced that the m6A modifications were highly enriched in the miR-380-3p, and deletion of METTL3 and METTL14 synergistically suppressed miR-380-3p expressions in the PC cells, hinting that m6A methylation is important to sustain the high-levels of miR-380-3p in PC. Interestingly, in addition to m6A modifications, other types of epigenetic modifications, such as m7G and m5C, are also reported to be involved in the regulation of miRNAs expressions [43][44][45]. However, the regulating effects of those modifications on miR-380-3p are still needed to be investigated in our future work.
Based on the previous data, miR-380-3p is deemed as an oncogene in cancers [29][30][31], which supports our results that miR-3803-p also played an oncogenic role in accelerating PC progression. Specifically, we evidenced that miR-380-3p positively regulated cell proliferation and division in vitro and tumorigenesis in vivo. In addition, epithelial-mesenchymal transition (EMT) is the first step for cancer cells to transform from noninvasive phenotypes into aggressive phenotypes [7,8], and early lymph node metastasis is considered as the main reason why PC patients are characterized with a high mortality rate [7,9]. Thus, blockage of EMT process is a novel strategy to hamper PC malignancy at early stage. Given that previous data suggest that miR-380-3p accelerates EMT process in breast cancer cells [31], we next asked whether miR-380-3p regulated EMT in PC cells in a similar manner. As expected, miR-380-3p suppressed E-cadherin, and upregulated N-cadherin and Vimentin to promote EMT in PC cells. The above data suggested that miR-380-3p acted as an oncogene to promote cell proliferation and EMT in PC cells.
MiRNAs (miRNAs) often exert their biological functions and affect cancer progression through targeting the 3ʹUTR of their downstream target genes, resulting in their degradation [38][39][40]. In consistent with this notion, multiple downstream targets, such as FOXO1 [28], SOX6 [46], and NLRP3 [47], can be targeted by miR-380-3p. In this study, we evidenced that miR-380-3p was capable of degrading PTEN mRNA by interacting with its 3ʹUTR. As previously reported, PTEN is a well-known tumor suppressor in various cancers, including PC [38][39][40], and upregulation of PTEN also suppresses cell proliferation and reverses the EMT process in cancers [38][39][40]. Interestingly, we evidenced that PTEN overexpression ablated the promoting effects of miR-380-3p overexpression on PC aggressiveness, suggesting that miR-380-3p PTEN-dependently accelerates PC aggressiveness. Also, based on the existing information, PTEN often exerts its tumor-inhibiting effects through inactivating the classical tumor-promoting Akt pathway in many types of cancer [41,42], which were validated by our study that miR-380-3p activated the Akt pathway in PC cells through degrading PTEN, resulting in the progression of PC.

Conclusions
Collectively, we conclude that METTL3 and METTL14-mediated m6A modifications were crucial for sustaining the high-expressed status of miR-380-3p in PC cells and tissues, and upregulation of miR-380-3p degraded PTEN to activate the tumor-promoting Akt pathway, resulting in the aggressiveness of PC. This study, for the first time, illustrated that m6A-dependent maturation and upregulation of miR-380-3p contributed to the development of PC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Consent to participate and for publication
All the co-authors agreed to publish the final version of this manuscript.

Authors' contributions
Zhijia Jiang and Xiaomeng Song are co-first authors, they are responsible for the conception, investigations, and manuscript drafting. Yaqing Wei, Yanxun Li, and Degang Kong are co-authors, they provide technical supports and help to collect and analyze the data. Jinjin Sun proofread and submitted this manuscript for publication.